A novel binding site between the voltage-dependent calcium channel Ca1.2 subunit and Caβ2 subunit discovered using a new analysis method for protein-protein interactions.

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (Ca1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 subunit using an epitope library of Ca1.2 resulted in two overlapping clones of the C-terminal sequence of Ca1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca1.2 and β2.